Small interfering RNA targeting Fas protects mice against renal ischemia-reperfusion injury

Abstract

Fas-mediated apoptosis has been suggested to contribute to tubular cell death after renal ischemia-reperfusion injury. Here we investigate whether small interfering RNA (siRNA) duplexes targeting Fas protect mice from acute renal failure after clamping of the renal artery. Renal ischemia-reperfusion injury was induced by clamping the renal vein and artery for 15 or 35 min. Mice were treated before or after ischemia with siRNA targeting Fas or a control gene, administered by hydrodynamic injection, low-volume renal vein injection, or both. Treated mice were evaluated for renal Fas protein and mRNA expression, tissue histopathology, and apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining. Blood urea nitrogen and survival were monitored in mice in which the contralateral kidney had been removed. A single hydrodynamic injection of Fas siRNA reduced Fas mRNA and protein expression in the kidney 4-fold. Kidneys from mice that received Fas siRNA two days earlier had substantially less renal tubular apoptosis by TUNEL staining and less tubular atrophy and hyaline damage. Whereas 90% of mice pretreated with saline or GFP siRNA died, only 20% of Fas-siRNA-pretreated animals died. The same survival advantage was provided by a single low-volume Fas siRNA injection into the renal vein. Moreover, postischemic injection through the renal vein protected 38% of mice from death. This study confirms the importance of Fas-mediated apoptosis in renal ischemia-reperfusion injury. Silencing Fas by systemic or local catheterization holds therapeutic promise to limit ischemia-reperfusion injury.

Fig. 1.

A single hydrodynamic injection of Fas siRNA silences Fas expression in kidneys subjected to 35 min of ischemia. (a) Silencing of Fas mRNA analyzed by RT-PCR of Fas and β-actin expression in kidney tissue homogenates from untreated mice or mice receiving a single hydrodynamic injection of PBS, Fas siRNA, or GFP siRNA. A representative sample is shown. These mice were not subjected to renal ischemia. However, similar results were obtained by real-time RT-PCR in injured mice (data not shown). (b and c) Silencing of Fas protein shown by immunohistochemistry (c). Mice injected 2 days earlier with PBS, Fas siRNA, or GFP siRNA were subjected to 35 min of renal ischemia and killed 1 day later. Control mice did not undergo ischemic insult. Graph (b) depicts percent Fas⁺ cells (mean and SD of all animals, three to five per group). (d) Reduced apoptosis in response to ischemia assessed by TUNEL staining. (Magnification, ×400.) (e) Graph of results from d. (f and g) Less cortical hyaline necrosis in mice pretreated with Fas siRNA compared with mice given GFP siRNA. (f) Representative hematoxylinand eosin-stained sections are shown. (Magnification, ×200.) (g) The histology score (mean, SD) was assessed in a blinded manner in groups of three to five mice subjected to ischemia (filled bars) or not (open bars).

Fig. 2.

Fas silencing after hydrodynamic and renal vein injection of Fas siRNA. Mice received a single hydrodynamic injection of Fas siRNA in PBS (filled bars) or just PBS (open bars) 4 days before and low-volume renal vein injection 2 days before the renal pedicle was clamped for 15 min (subcritical ischemia) or not in sham-operated animals. Samples were harvested 1 or 2 days after clamping, as indicated. (a) BUN increased when the right kidney was removed in control animals but not in Fas-siRNA-treated mice. (b) Ischemia-induced up-regulation of Fas mRNA, compared with GAPDH mRNA by real-time RT-PCR, was largely silenced in animals receiving a single hydrodynamic injection of Fas siRNA. (c and d) Fas protein up-regulation was also blunted in Fas-siRNA-treated mice. (c) Representative sections. (Magnification, ×200.) (d) Fas staining of the ischemic left kidney and the unclamped right kidney (mean and SD). (e) The proportion of TUNEL⁺ tubules was also reduced by Fas siRNA treatment.

Fig. 3.

Hydrodynamic or renal vein injection of Fas siRNA protects mice from lethal kidney ischemia: survival and BUN levels of surviving mice after 35 min of kidney ischemia and reperfusion. (a) Mice were treated with saline (blue) (n = 5), GFP siRNA (green) (n = 5), or Fas siRNA (red) (n = 10) by hydrodynamic injection (♦), renal vein injection (▴), or both (▪). (b) BUN is reduced in surviving Fas-siRNA-treated mice (filled bars) compared with saline-treated controls (open bars). (c) Intraoperative (postischemia) treatment via the renal vein also offered some protection (color code as in a).